Nontypeable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, pneumonia, and bronchitis. Following each episode of acute otitis media, fluid remains in the middle ear for weeks to months and is associated with significant hearing deficit, which in turn can impair language acquisition, speech development, and school performance. The initial step in the pathogenesis of disease due to nontypeable H. influenzae involves colonization of the upper respiratory epithelium. We have demonstrated that two related high-molecular-weight proteins called HMW1 and HMW2, which are present in 75 - 80% of all nontypeable H. influenzae strains, promote attachment to human epithelium. Based on our in vitro results, we speculate that these proteins are important colonization factors. In recent work, we have established that the HMW1 and HMW2 proteins are localized on the surface of the organism by a mechanism that has novel features compared with known bacterial secretion systems. In addition, we have discovered that expression of HMW1 and HMW2 is phase variable, enabling the organism to adapt to diverse environments and evade the host immune response. In the present proposal we plan to elucidate the molecular details of the pathway by which HMW1 and HMW2 are presented on the bacterial surface. In particular, we will dissect the influence of an unusual N-terminal signal sequence, determine the function of 373 amino acid fragment that is ultimately cleaved, and characterize the roles of accessory proteins called HMWB and HMWC. In additional experiments, we will elucidate the mechanism by which expression of HMW1 and HMW2 is regulated, focusing in particular on the function of 7-base pair tandem repeats that lie within the promoters of the HMW1 and HMW2 structural genes and undergo spontaneous variation in number. We will examine the role of a conserved 19-base pair sequence that is upstream of the repeats in the promoters of the HMW1 and HMW2 structural genes and upstream of the hmwB and hmwC genes. From a practical perspective, the results of these studies may be directly relevant to the development of novel antimicrobials effective in the treatment and prevention of nontypeable H. influenzae disease. More generally, they may provide fundamental insights into host-microbial relationship, protein secretion and gene regulation.